Maternal obesity increases the risk of hepatocellular carcinoma through the transmission of an altered gut microbiome

Background & Aims Emerging evidence suggests that maternal obesity negatively impacts the health of offspring. Additionally, obesity is a risk factor for hepatocellular carcinoma (HCC). Our study aims to investigate the impact of maternal obesity on the risk for HCC development in offspring and elucidate the underlying transmission mechanisms. Methods Female mice were fed either a high-fat diet (HFD) or a normal diet (ND). All offspring received a ND after weaning. We studied liver histology and tumor load in a N-diethylnitrosamine (DEN)-induced HCC mouse model. Results Maternal obesity induced a distinguishable shift in gut microbial composition. At 40 weeks, female offspring of HFD-fed mothers (HFD offspring) were more likely to develop steatosis (9.43% vs. 3.09%, p = 0.0023) and fibrosis (3.75% vs. 2.70%, p = 0.039), as well as exhibiting an increased number of inflammatory infiltrates (4.8 vs. 1.0, p = 0.018) and higher expression of genes involved in fibrosis and inflammation, compared to offspring of ND-fed mothers (ND offspring). A higher proportion of HFD offspring developed liver tumors after DEN induction (79.8% vs. 37.5%, p = 0.0084) with a higher mean tumor volume (234 vs. 3 μm3, p = 0.0041). HFD offspring had a significantly less diverse microbiota than ND offspring (Shannon index 2.56 vs. 2.92, p = 0.0089), which was rescued through co-housing. In the principal component analysis, the microbiota profile of co-housed animals clustered together, regardless of maternal diet. Co-housing of HFD offspring with ND offspring normalized their tumor load. Conclusions Maternal obesity increases female offspring’s susceptibility to HCC. The transmission of an altered gut microbiome plays an important role in this predisposition. Impact and implications The worldwide incidence of obesity is constantly rising, with more and more children born to obese mothers. In this study, we investigate the impact of maternal diet on gut microbiome composition and its role in liver cancer development in offspring. We found that mice born to mothers with a high-fat diet inherited a less diverse gut microbiome, presented chronic liver injury and an increased risk of developing liver cancer. Co-housing offspring from normal diet- and high-fat diet-fed mothers restored the gut microbiome and, remarkably, normalized the risk of developing liver cancer. The implementation of microbial screening and restoration of microbial diversity holds promise in helping to identify and treat individuals at risk to prevent harm for future generations.


Supplementary materials and methods
Transgenic HCC mouse model LAP-tTA (LAP, B6.NMRI-Tg(Cebpb-tTA)5Bjd/Cnbc, EM:04498) x TRE MYC (B6.Cg-Tg(tetO-MYC,-OVAL)#Gtgm/Ieg, EM:04319) (LAP-Myc) mice were used as HCC transgenic mouse model.Single transgenic LAP-tTA mothers received a HFD for twelve weeks before mating with lean TRE MYC males.Myc expression in the liver was activated by removing the doxycycline enriched diet (Safe Diet, 0.625g/Kg Doxycycline Hyclate) [1,2].Offspring were assigned to one of the following groups: Stop of doxycycline at 5 weeks with feeding of normal diet (ND) (Dox5w_F1ND), stop of doxycycline at 5 weeks with feeding of choline-deficient methionine controlled high-fat diet (MCD) (Dox5w_F1MCD), or stop of doxycycline at 3 weeks of age with feeding of a ND (Dox3w_F1ND).All animals were sacrificed at 16 weeks.

Physiological and metabolic phenotyping
Body composition (fat and fat-free mass) was determined in vivo using quantitative magnetic resonance (EchoMRI™ 3-in-1 v2.1; Echo Medical Systems, Houston, TX) at different time points.Glycemia was determined using an Accu-Check glucometer (Roche Diabetes Care, Rotkreuz, Switzerland).For fasting glycemia, mice were fasted for 6 hours.

Liver histopathological analysis and immuno-labelling
Liver samples were processed routinely in 10% neutral buffered formalin for 24 h, and then embedded in paraffin.The fixed liver tissues sliced into 5-μm thickness and stained with haematoxylin and eosin (H&E) and Masson Trichrome (MT) to evaluate liver histology, tumor histology and grading of liver injury.Leukocytes, Macrophages and CD8 T cells were labelled with an anti-CD45 (clone D3F8Q), an anti-Iba1 (clone EPR16588) or an anti-CD8 (clone D4W2Z) followed by an Alexa488-goat anti-rabbit IgG (A32731, Invitrogen) or the SignalStain® Boost IHC Detection Reagent (HRP anti-rabbit, Cell Signaling Technology).
Sagittal sections of the median and the left liver lobe (one each, per animal) and axial sections of intestines (four per animal) were included in the analysis.Whole slide images were scanned with the Zeiss Axioscan-Z1 Microscope Slide Scanner (Carl Zeiss AG, Jena, Germany).Parts of the slide containing tissue were detected in QuPath using the SimpleTissueDetection function.The thereby created annotations were automatically assessed with the createAnnotationFromPixelClassifier function using a trained pixel classifier to only include liver tissue without large vessels or preparation artefacts.
For steatosis, lipid vacuoles were automatically detected and validated visually in H&E stained slides.The relative area of steatosis was calculated as the area of vacuoles normalized by the total area of the whole slide.
Fibrosis, stained in blue in MT slides was automatically quantified using a manually trained pixel classifier, which discriminates fibrosis based on pixel coloration.By applying this threshold, we determined the percentage of fibrosis on the total surface of each section.

Assessment of intestinal histopathology
The count of Paneth cells was visually conducted on 50 villi per section.Only cells with a clearly visible nucleus and positive staining (lysozyme) throughout the cytoplasm were considered.By dividing the number of counted Paneth cells by the number of vili, the Paneth cell/villus ratio was obtained.This was performed on three sections per specimen.The count of lymphatic vessels (Lyve-1 labelling) was also visually conducted around the entire circumference of the sections.Only lymphatic vessels with a visible lumen and present in the submucosa were counted.By dividing the number of lymphatic vessels by the total number of villi, the lymphatic vessel/villus ratio was obtained.This was performed on three sections per specimen.

Hepatic gene expression
After total RNA extraction of liver tissue (ReliaPrepTM RNA Tissue, Promega, Madison WI, USA), cDNA was synthesised by extending a mix of random primers with the High Capacity cDNA Reverse Transcription Kit in the presence of RNase inhibitor (Applied Biosystems).The relative quantity of each transcript was normalized to the expression of Eef1, Hprt, and Gapdh.SYBRGreen reagent was used for Real-time PCR on the ABI Prism 7000 sequence detection system (Applied Biosystems, Waltham MA, United States) according to the manufacturer's instructions.Primer sequences are provided in the Supplementary CTAT Table .Reconstruction of 3D tumor volume 3D reconstructions were performed with liver and bone segmentation via the Imalytics software (Imalytics Preclinical 3.0 (Gremse-IT GmbH, Aachen, Germany).
3D rendering was performed after tumor morphological identification by manually circling ROI in 1mm thick, three axial slice views.

Fig. S1 .
Fig. S1.Maternal obesity does not affect steatosis before adulthood and increases compensatory proliferation Female and male offspring of obese (HFD_Fem & HFD_Mal) and lean mothers (ND_Fem & ND_Mal) were fed a normal diet.(A) Histological hematoxylin-eosin stained sections from male offspring at 3 weeks of age, as well as from female and male offspring at 8 weeks of age from ND and HFD mothers.(B) Automated quantification of steatosis, expressed as % of liver surface covered by steatotic vesicles.(C) Histological sections with immunohistochemistry staining of cleaved caspase 3, (D) Ki67 in immune cells, and (E) Ki67 in hepatocytes between the two groups with corresponding quantification of stained area.Data presented as median ± IQR, one dot represents one animal, level of significance of p=0.05.Statistical analysis was performed by Wilcoxon-Mann-Whitney test (B, C, D, E).ND_Fem: Female offspring born to lean mothers n = 6-15, F_HFD: Female offspring born to obese mothers n = 6-10, M_ND male offspring born to lean mothers n = 5-7, M_HFD male offspring born to obese mothers n = 5-8.Scale bar Panel A: 100µm, Panels C-E: 50µm.

Fig. S2 .
Fig. S2.Maternal obesity does not affect intestinal inflammation and the gut vascular barrier Female and male offspring of obese (HFD_Fem & HFD_Mal) and lean mothers (ND_Fem & ND_Mal) were fed a normal diet.(A) Axial histological sections of intestines with Immunohistochemistry staining for lymphatic vessels with anti-LYVE-1.Manual quantification of lymph vessels per crypt (right).(B) Axial histological sections of intestines with Immunohistochemistry staining for Paneth cells with anti-lysozyme.Manual quantification of Paneth cells per crypt (right).(C) Axial histological sections of intestines immunohistochemistry staining for gut vascular barrier with anti-PV-1.Automated quantification of stained area (right).Data presented as median ± IQR, one dot represents one animal, level of significance of p=0.05.Statistical analysis was performed by Wilcoxon-Mann-Whitney test (A, B, C).ND_Fem: Female offspring born to lean mothers n = 8, F_HFD: Female offspring born to obese mothers n = 7, M_ND male offspring born to lean mothers n = 7, M_HFD male offspring born to obese mothers n = 9.Scale bar: 100µm.

Fig. S3 .
Fig. S3.Maternal obesity does not influence the risk to develop HCC in a shortterm transgenic HCC model (A) HCC carcinogenesis in double transgenic offspring (LAP-tTA x TRE-MYC) of obese or lean mothers was induced through removal of doxycycline.Offspring were assigned to one of the following groups: Stop of doxycycline at 5 weeks with feeding of normal diet (ND) (Dox5w_F1ND), stop of doxycycline at 5 weeks with feeding of methionine choline deficient diet (MCD) (Dox5w_F1MCD), or stop of doxycycline at 3 weeks of age with feeding of a ND (Dox3w_F1ND) All animals were sacrificed at 16 weeks.(B) The proportion of animals that developed 0, 1-2 or more than 2 tumors for all groups on the left or per group on the right.(C) The total number of tumors per animal and (D) the total tumor volume per animal.(E) The total tumor volume or number differentiated by diet of the mother and sex of the offspring.Data presented as median ± IQR, one dot represents one animal, level of significance of p=0.05.Statistical analysis was performed by Wilcoxon-Mann-Whitney test (C, D, E).Median with interquartile ranges.Dox5w_F1ND n=23 (ND=11 and HFD=12), Dox5w_F1MCD n=33 (ND=12, HFD=21, Dox3w_F1ND n=24 (ND=11, HFD=13).

Fig. S4 .
Fig. S4.Expression of inflammatory markers or Toll-like receptor family members in DEN treated offspring Offspring of obese and lean mothers were either separately housed (ND_Sep and HFD_Sep) or co-housed (ND_CoH and HFD_CoH) after weaning.(A) Real-time PCR expression analysis of inflammatory markers such as Monocyte Chemoattractant Protein-1 (MCP-1), inducible Nitric Oxide Synthase (iNOS), Class II Major Histocompatibility Complex (MHC II) molecules, and chemokine CXCL16.Expression of Toll-like Receptor (TLR) family member 2 and 4. Data presented as median ± IQR, one dot represents one animal, level of significance of p=0.05.Statistical analysis was performed by Wilcoxon-Mann-Whitney test.ND_Sep: Female offspring born to lean mothers housed seperately n = 10, HFD_Sep: Female offspring born to obese mothers housed separately n = 10, ND_CoH female offspring born to lean mothers co-housed with offspring of obese mothers n = 10, HFD_CoH female offspring born to obese mothers cohoused with offspring of lean mothers n = 10